tRNA Processing

tRNA处理

• 批准号: 10536625
• 负责人: Eric M. Phizicky
• 金额: $ 44.41万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536625
• 关键词: 7-methylguanosine; Address; Adenosine; Amino Acids; Amino Acyl-tRNA Synthetases; Anticodon; Binding; Biology; Cell Nucleus; Charge; Coupled; Cytoplasm; Defect; Distant; Eukaryota; Exons; Exonuclease; Exposure to; Fission Yeast; Gene Expression; Genetic; Growth; Health; Human; Impairment; Intellectual functioning disability; Introns; Measures; Messenger RNA; Methods; Methylation; Mitochondrial Diseases; Modification; Molecular Biology; Monitor; Mutation; Nuclear; Organism; Orthologous Gene; Pathway interactions; Peptides; Phenylalanine-Specific tRNA; Phosphotransferases; Post-Translational Protein Processing; Process; Proline-Specific tRNA; Property; RNA Decay; RNA Processing; RNA Splicing; RNA Stability; Rattus; Ribosomes; Role; Saccharomyces cerevisiae; Source; Structure; Temperature; Testing; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; X-linked mental retardation 9; Yeasts; exosome; follow-up; mutant; nervous system disorder; novel; nuclease; prevent; programs; response; restoration; spleen exonuclease; stem; temperature sensitive mutant; transcriptome sequencing

ABSTRACT tRNAs are highly evolved in all organisms for specific recognition by cognate tRNA synthetases, high fidelity decoding, efficient use in translation, and high stability. The ubiquitous tRNA modifications are highly conserved in eukaryotes, and many have crucial roles in the yeast Saccharomyces cerevisiae and in human health. Modifications in the tRNA body (outside the anticodon loop) are crucial for tRNA stability in yeast, and associated with several neurological disorders in humans. We study the rapid tRNA decay (RTD) pathway in S. cerevisiae, which targets a subset of mature tRNAs lacking any of several body modifications, due to exposure of the 5' end to the 5'-3' exonucleases Rat1 and Xrn1. RTD also frequently occurs in tRNA variants with destabilizing mutations exposing the 5' end, and is inhibited in met22Δ mutants due to increased levels of adenosine 3',5' bis-phosphate (pAp) and its inhibition of Rat1 and Xrn1. Little is known about RTD or the biology of body modifications in any other eukaryote. To address this, we are studying these processes in the fission yeast Schizosaccharomyces pombe because of its ~600 million years evolutionary distance from S. cerevisiae, and because of its facile genetics and molecular biology. We have recently uncovered an unusual decay pathway in S. cerevisiae in which pre-tRNAs are degraded in the cytoplasm by a pathway regulated by Met22. This Met22-regulated pre-tRNA decay (MPD) pathway is independent of RTD, because unlike classical RTD, it does not require the Rat1 or Xrn1 exonucleases and does not act on mature tRNA, and it is novel because it is also independent of the nuclear surveillance tRNA decay pathway, which acts in the nucleus on pre-tRNAs through Trf4, RRP6 and the nuclear exosome. Rather, MPD occurs on unspliced pre-tRNA that accumulates in the cytoplasm due to impaired intron-exon structure. We also study modifications in the anticodon loop, due to their importance in translation, with a focus on Trm7, which 2’-O-methylates N32 and N34 in the anticodon loop of certain tRNAs. S. cerevisiae and S. pombe trm7 mutants have severe growth defects, while humans with mutations have intellectual disability. Our prior results showed that the growth defect of S. cerevisiae and S. pombe trm7 mutants was due to reduced function, but not reduced amounts, of tRNAPhe. We recently discovered an unusual property of S. cerevisiae and S. pombe trm7Δ mutants: each mutant robustly activates the general amino acid control (GAAC) response, which massively reprograms gene expression in all eukaryotes due to uncharged tRNA sensed by Gcn2 kinase, but trm7Δ mutants do not exhibit a detectable tRNA charging defect. To follow up, we will: 1) Examine similarities and differences in the RTD pathway and body modification biology in S. pombe 2) Define how Met22-regulated pre-tRNA decay of anticodon stem variants occurs in S. cerevisiae 3) Define how trm7Δ mutants activate the GAAC pathway and how Trm7 recognizes tRNAs.

抽象的 在所有生物体中，TRNA都高度进化，以通过同源tRNA合成酶特异性识别，高 保真度解码，在翻译中有效使用和高稳定性。无处不在的tRNA修饰高度 在真核生物中保守，许多在酵母糖酵母和人类中具有至关重要的作用 健康。 tRNA体的修饰（在反密码子循环外）对于酵母中的tRNA稳定性至关重要，并且 与人类的几种神经系统疾病有关。我们研究了S. Cerevisiae，靶向由于暴露于几种身体修饰中的任何一个成熟的TRNA的子集 5'-3'外丝线的5'末端Rat1和Xrn1。 RTD也经常发生在tRNA变体中 破坏稳定的突变暴露了5'端，由于水平升高而被抑制在Met22δ突变体中 腺苷3'，5'双磷酸（PAP）及其对Rat1和Xrn1的抑制作用。 关于RTD或任何其他真核生物中身体修饰的生物学知之甚少。为了解决这个问题，我们 正在研究这些过程在裂变酵母菌酸果实中，因为它的6亿 与酿酒酵母的进化距离，以及其易于遗传学和分子生物学。 我们最近在酿酒酵母中发现了一种不寻常的衰减途径，其中pre-trnas被降解 在细胞质中，由MET22调节的途径。该MET22调节的前型型衰减（MPD）途径为 独立于RTD，因为与经典RTD不同，它不需要RAT1或XRN1外丝线和 不对成熟的tRNA作用，它是新颖的，因为它也独立于核监视tRNA 通过TRF4，RRP6和核外泌体在TRNA上作用于核的衰减途径。相当， MPD发生在由于内含子 - 外激素结构受损而导致细胞质中积聚的未填充的pre-tRNA上。 我们还研究了反登陆循环中的修改，因为它们在翻译中的重要性，重点是 TRM7，在某些TRNA的反密码子环中的2'-O-甲基化N32和N34。 S. cerevisiae和S.pombé TRM7突变体具有严重的生长缺陷，而具有突变的人具有智力障碍。我们的先验 结果表明，酿酒酵母和Pombe链球菌TRM7突变体的生长缺陷是由于降低 功能，但不会减少trnaphe。我们最近发现了酿酒酵母的不寻常特性 和S. pombeTRM7δ突变体：每个突变体可鲁棒地激活一般的氨基酸对照（GAAC） 响应，由于未充电的tRNA所感受到的所有真核生物中的基因表达 GCN2激酶，但TRM7Δ突变体不存在可检测的tRNA充电缺陷。 为了跟进，我们将：1）检查RTD途径和身体修改的相似性和差异 S. pombe中的生物学2）定义了Met22调节的反密码子茎变体的pre-tRNA衰变。 酿酒酵母3）定义TRM7δ突变体如何激活GAAC途径以及TRM7如何识别TRNA。